We seek to understand the impact that oxidative stress has upon organisms by addressing three overarching questions: How are reactive oxygen species formed in biological environments? What biomolecules do they damage? And how do cells defend themselves against them? We have employed E. coli as a model organism, in part because we have such a detailed understanding of its physiology and biochemistry, and in part because this facultative anaerobe can be genetically manipulated in the absence of oxygen. Our next set of specific aims addresses each of these three issues: 1. Are cytoplasmic enzymes shielded from H202 that is generated by periplasmic enzymes? Is intracellular formation of reactive oxygen species especially rapid inside aerated anaerobes? Are these species the primary factor in blocking their aerobic growth? Do antibiotics actually trigger H202 stress? 2. Does superoxide damage mononuclear iron enzymes ? Do oxidants inhibit ferrochelatase? What other targets of these oxidants can we discover through transcriptome analysis? 3. How completely can manganese-fed E. coli dispense with iron? How efficiently is manganese delivered to metalloenzymes? Do lactic-acid bacteria that routinely experience H202 stress employ manganese, rather than iron, in mononuclear non-redox enzymes? What about obligate aerobes, such as eukaryotes? These questions follow directly from the results of the current project. They are fundamental to our understanding of oxidative stress in contexts of medical interest, including the pathogenicity of obligate anaerobes, the effectiveness of antibiotic and antitumor treatments, and the mechanisms by which phagocytes suppress microbial infections.